Fig 2: DNMT1 is stabilized in TOPORS-KO cells.a Effects of TOPORS and TOPORS-RD add-back on TOPORS-KO MDS-L cell growth in the presence and absence of 12.5 nM DAC (n = 3 for each group, biological replicates). Reduction rate of cell numbers on day 7 of culture compared to those of WT MDS-L cells are depicted (right). Full-length TOPORS with silent mutations (CGC to AGA) in the sgTOPORS#1 target sequence was used. b Outline of the second CRISPR-Cas9 screening using TOPORS-KO single-cell clones with and without DAC. c Scatter plots showing the β-scores of each gene in the presence and absence of DAC. Candidate genes with proportionally increased sgRNA read counts during culture in the presence of DAC compared to those in the absence of DAC are indicated by red and purple dots. d Venn diagram showing the two overlapping candidate genes, UHRF1 and DNMT1, between MOLM-13 and MDS-L screenings. e Changes in DNMT1 and UHRF1 protein levels after exposure to low-dose DAC (12.5 nM) in WT and TOPORS-KO MDS-L cells. Unmodified (main band) and modified (sub-band) DNMT1 are indicated. The samples derive from the same experiment but different gels for DNMT1, β-actin, and another for UHRF1 were processed in parallel. f DAC-induced SUMOylation and degradation of DNMT1. WT and TOPORS-KO MDS-L cells were exposed to a high dose of DAC (10 µM) for 8 h in the presence and absence of the SUMO inhibitor, ML-792 (3 µM), or the proteasome inhibitor, MG132 (20 µM). Unmodified (main band) and modified (sub-band) DNMT1 are indicated. Changes in DNMT1 protein levels after exposure to high-dose DAC (10 µM) for 8 h in WT and TOPORS-KO MOLM-13 cells (g) and mouse MLL-AF9 leukemic cells (h). i Reduction rate of DNMT1 unmodified main bands after DAC treatment. Intensity of DNMT1 main bands after DAC treatment in (g, h, and i) relative to those before DAC treatment are indicated (n = 3 for each group, independent replicates). **p < 0.01; ***p < 0.001; n.s., not significant by unpaired two-tailed Student’s t-test. Data are presented as mean ± SD. Source data are provided as a Source Data file.

Fig 3: Topors is largely dispensable for normal hematopoiesis.a Strategy for making a knockout allele for Topors by homologous recombination in embryonic stem cells. A large portion of exon 3 encoding amino acids 67 to 782 was replaced by a neo-cassette. b Representative PCR-based genotyping of mice obtained from breeding heterozygotes. Tail DNA was used as a template. Complete blood counts (c), proportions of myeloid cells (My; Mac-1+ and/or Gr-1+), B220+ B cells, and CD4+ or CD8+ T cells in PB (d), and proportions of BM HSPCs (e) in WT and Topors−/− mice (2–4 months old) (n = 5 for each group, biological replicates). LSK Lin−Sca-1+c-Kit+; MP myeloid progenitor; CMP common myeloid progenitor; GMP granulocyte/macrophage progenitor; MEP megakaryocyte/erythrocyte progenitor. f Transplantation assays. Total BM cells (5 × 106) from WT (red) and Topors−/− (blue) mice were transplanted into lethally irradiated CD45.1 recipient mice. Complete blood counts and proportions of myeloid, B, and T cells in PB at 6 months post-transplantation are depicted (n = 6, biological replicates). g Growth of WT and Topors−/− MLL-AF9-transformed cells in the presence and absence of DAC (n = 3 for each group, biological replicates). h Growth of WT and Topors−/− c-Kit-positive cells in the presence and absence of DAC (n = 3 for each group, biological replicates). i In vivo treatment of MLL-AF9 leukemic cells with DAC. WT and Topors−/− MLL-AF9 leukemic cells were transplanted into lethally irradiated recipient mice with support BM cells from CD45.1 mice. The recipient mice were treated with DAC from 4 to 6 weeks after transplantation. Chimerism of CD45.2 leukemic cells (left panels) and their absolute numbers (right panels) in PB at 4 and 6 weeks. The values of chimerism and absolute numbers at 6 weeks relative to those at 4 weeks (ratio) are also plotted, respectively (n = 9 for WT group, n = 10 for Topors−/− group, biological replicates). *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant by unpaired two-tailed Student’s t-test. Data are presented as mean ± SD. Source data are provided as a Source Data file.

Fig 4: CRISPR-Cas9 screening reveals that TOPORS KO augments sensitivity to HMAs.a Outline of CRISPR-Cas9 screening with and without HMAs. Screening results using MOLM-13 cells treated with DAC at 20 nM, (b) and AZA at 250 nM, (c) and MDS-L cells treated with DAC at 12.5 nM, (d) and AZA at 100 nM (e). Scatter plots showing the β scores of each gene, which indicate changes in the sgRNA content for each gene, in the presence and absence of HMAs. Candidate genes whose sgRNAs decreased during culture specifically in the presence of HMAs are indicated by blue dots (DAC) and red dots (AZA). f List of candidate genes identified to be in common in the presence of DAC and AZA. The p-value was calculated using a two-sided test. g Venn diagrams comparing screening results and the list of candidate genes identified to be in common in the presence of DAC and AZA. (h) Schematic representation of the TOPORS protein with the RING finger domain, the SUMO-1 interacting domain, and putative SUMO-interaction motifs (SIMs). Source data are provided as a Source Data file.

Fig 5: TOPORS-KO MDS/AML cells show increased apoptosis and mitotic defects upon DAC treatment.a Frequency of apoptotic cell death in WT and TOPORS-KO MDS-L cells after exposure to 12.5 nM of DAC. Representative flow cytometric profiles of cells at 72 h of DAC exposure (left panel). Percentage of Annexin V-positive cells (n = 3 for each group, technical replicates, the experiments were repeated twice independently) (right panel). b Cell cycle status of WT and TOPORS-KO MDS-L cells after exposure to 12.5 nM of DAC evaluated by EdU incorporation and DAPI staining. Representative flow cytometric profiles of cells at 72 h of DAC exposure (left panel). Proportion of each cell cycle (middle panel) and hyperploidy cells at the indicated time points (right panel) (n = 3 for each group, technical replicates, the experiments were repeated twice independently). Data are presented as mean ± SD. Source data are provided as a Source Data file.